Identification of LATS1 Phosphorylation Motifs

Thirty minutes after release from nocodazole-induced prometaphase arrest, HeLa cells were collected and immunoprecipitated with an anti-CDC27 antibody. In vitro kinase reactions were then performed using the immunoprecipitates and cold ATP. The resulting samples were analyzed by nano-liquid chromatography/tandem mass spectrometry, as described previously [10 (link)]. The LATS1 phosphorylation motifs were identified using in vitro kinase assays, as described previously [38 (link)]. Briefly, HeLa cell lysates were dephosphorylated with thermo-sensitive alkaline phosphatase (Promega). After heat inactivation of the phosphatase, the lysates were diluted with 40 mM Tris-HCl (pH 7.5) containing 20 mM MgCl₂ and 1 mM ATP, and then reacted with recombinant LATS1 for 3 h at 37°C. The reaction mixture was denatured with 8 M urea followed by reductive alkylation and tryptic digestion. The phosphopeptides were enriched with hydroxyl acid-modified metal oxide chromatography [18 (link)] using titania and analyzed via nano-liquid chromatography/tandem mass spectrometry. Based on the LATS1 substrate sites identified in vitro, phosphorylation motifs were obtained using motif-x [39 (link)].